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Introduction to Alignment | Griffith Lab

RNA-seq Bioinformatics

Introduction to bioinformatics for RNA sequence analysis

Introduction to Alignment

Course

Module 2 - Key concepts

  • Splice-aware alignment, SAM/BAM format, SAM flags, CIGAR strings, SAM sorting, etc.

Module 2 - Learning objectives

  • RNA-seq alignment challenges and common questions
  • Alignment strategies
  • HISAT2
  • Introduction to the BAM and BED formats
  • Basic manipulation of BAMs
  • Visualization of RNA-seq alignments in IGV
  • Alignment QC Assessment
  • BAM read counting and determination of variant allele expression status

Lectures

Course
Pre-alignment QC | Griffith Lab

RNA-seq Bioinformatics

Introduction to bioinformatics for RNA sequence analysis

Pre-alignment QC

Course

RNA-seq_Flowchart

FastQC

You can use FastQC to get a sense of your data quality before alignment:

Video Tutorial here:

Try to run FastQC on your fastq files:

cd $RNA_HOME/data
fastqc *.fastq.gz

Then, go to the following url in your browser:

Exercise: Investigate the source/explanation for over-represented sequences:

Fastp

Fastp is a similar alternative tool. QC results for this tool can be produced as follows

cd $RNA_HOME/data
mkdir fastp
cd fastp
mkdir HBR_Rep1 HBR_Rep2 HBR_Rep3 UHR_Rep1 UHR_Rep2 UHR_Rep3

cd $RNA_HOME/data/fastp/HBR_Rep1
fastp -i $RNA_HOME/data/HBR_Rep1_ERCC-Mix2_Build37-ErccTranscripts-chr22.read1.fastq.gz -I $RNA_HOME/data/HBR_Rep1_ERCC-Mix2_Build37-ErccTranscripts-chr22.read2.fastq.gz

cd $RNA_HOME/data/fastp/HBR_Rep2
fastp -i $RNA_HOME/data/HBR_Rep2_ERCC-Mix2_Build37-ErccTranscripts-chr22.read1.fastq.gz -I $RNA_HOME/data/HBR_Rep2_ERCC-Mix2_Build37-ErccTranscripts-chr22.read2.fastq.gz

cd $RNA_HOME/data/fastp/HBR_Rep3
fastp -i $RNA_HOME/data/HBR_Rep3_ERCC-Mix2_Build37-ErccTranscripts-chr22.read1.fastq.gz -I $RNA_HOME/data/HBR_Rep3_ERCC-Mix2_Build37-ErccTranscripts-chr22.read2.fastq.gz

cd $RNA_HOME/data/fastp/UHR_Rep1
fastp -i $RNA_HOME/data/UHR_Rep1_ERCC-Mix1_Build37-ErccTranscripts-chr22.read1.fastq.gz -I $RNA_HOME/data/UHR_Rep1_ERCC-Mix1_Build37-ErccTranscripts-chr22.read2.fastq.gz

cd $RNA_HOME/data/fastp/UHR_Rep2
fastp -i $RNA_HOME/data/UHR_Rep2_ERCC-Mix1_Build37-ErccTranscripts-chr22.read1.fastq.gz -I $RNA_HOME/data/UHR_Rep2_ERCC-Mix1_Build37-ErccTranscripts-chr22.read2.fastq.gz

cd $RNA_HOME/data/fastp/UHR_Rep3
fastp -i $RNA_HOME/data/UHR_Rep3_ERCC-Mix1_Build37-ErccTranscripts-chr22.read1.fastq.gz -I $RNA_HOME/data/UHR_Rep3_ERCC-Mix1_Build37-ErccTranscripts-chr22.read2.fastq.gz

MultiQC

Run MultiQC on your fastqc reports to generate a single summary report across all samples/replicates.

cd $RNA_HOME/data
multiqc ./

Then, go to the following url in your browser:

Clean up

Move all the FASTQC files into their own directory

cd $RNA_HOME/data
mkdir fastqc
mv *_fastqc* fastqc

PRACTICAL EXERCISE 4

Assignment: Run FASTQC on one of the additional fastq files you downloaded in the previous practical exercise.

Run FASTQC on the file ‘hcc1395_normal_1.fastq.gz’ and answer these questions by examining the output.

Questions

Solution: When you are ready you can check your approach against the Solutions.

Course